Automated method of calibrating a chromatography system and analysis of a sample

ABSTRACT

An automated method of calibrating a chromatography system and analyzing a sample is described. The method includes forming diluted standard solutions that are injected into a chromatography column. The detected peaks can be identified based on a first predetermined calibration ratio associated with the standard solution. Once the chromatography system is calibrated, samples can be chromatographically analyzed where the measured peaks are identified and quantified in an automated manner.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional application and claims the priority benefit ofpending application Ser. No. 14/490,527 filed on Sep. 18, 2014, thedisclosure of which is incorporated herein by reference.

BACKGROUND

Chromatography is a widely used analytical technique for the chemicalanalysis and separation of molecules. Chromatography involves theseparation of one or more analyte species from matrix components presentin a sample. The analytes and matrix components have an affinity for thestationary phase. In ion exchange chromatography, the stationary phaseincludes ionic moieties that ideally will bind to the charged analyteswith varying levels of affinity. An eluent is percolated through thestationary phase and competes with the analyte and any matrix componentsfor binding to the ionic moieties. The eluent is a term used to describea liquid or buffer solution that is pumped into a chromatography column.During this competition, the analyte and any matrix components willseparate from each other by eluting off of the stationary phase as afunction of time and then be subsequently detected at a detector.Examples of some typical detectors are a conductivity detector, a UV-VISspectrophotometer, and a mass spectrometer.

Chromatography typically requires a calibration process to both identifythe analyte of interest and quantitate the amount of analyte. A standardsolution is often used as part of a calibration process for determiningthe chemical identity of the chromatographic peaks of a sample solution.The standard solution can have one or more types of analytes where eachone is at a known predetermined concentration. A chromatogram of thestandard solution will provide the retention times, peak heights, andpeak areas for the analytes in the standard solution. Such informationacquired with the standard solution can be compared to the chromatogramof a sample solution to determine the chemical identity andconcentration of the components in the sample solution. For instance,the peak retention times of the standard solution can be correlated withthe peak retention time of the sample solution to determine the chemicalidentity of the peaks of the sample chromatogram. The chemicalidentities of the standard components are verified by injectingindividual pure components of the standard and these identities areestablished by the peak retention times.

In regards to quantitation, several dilutions of the standard solutioncan be prepared manually and analyzed chromatographically. It should benoted that user error can be significant when manually preparing dilutedstandard solutions. The measured peak areas or peak heights from thediluted standards can then be used to calculate calibration slopes andintercepts for each of the analytes in the standard solution. A samplecontaining one or more analytes can be analyzed chromatographically tomeasure one or more peak areas corresponding to analytes. These peakareas can be used to quantitate analyte concentrations based on thecorresponding calibration slopes and intercepts. Applicant believes thatthere are several problems with calibration and sample analysisprocesses in that the process needs to be more automated in analyteidentification, diluting standard solution for calibration with reduceduser input, determining proper calibration concentration ranges, andautomated sample concentration calculations.

SUMMARY

In a first method embodiment of calibrating a chromatography system, itincludes an automatic identification step based on a first predeterminedcalibrant ratio and another automatic identification step based on peakretention times, and a first and second analyte time intervals. Thisfirst method is well suited for calibrating analyte ions that arestrongly dissociated, weakly dissociated, and combinations thereof.

The first method includes forming a first diluted standard solution bymixing a standard material and a diluent. The first diluted standardsolution includes a first analyte having a first diluted calibrantconcentration, and a second analyte having a second diluted calibrantconcentration, in which the first diluted standard solution isconfigured to have a first predetermined calibrant ratio. The firstdiluted standard solution is injected into a chromatographic separator.The first diluted standard solution is separated in the chromatographicseparator. A first peak and a second peak are measured with a detector.The first peak and second peak have a first retention time and a secondretention time, respectively. Automatically identifying whether thefirst peak corresponds to the first analyte or the second analyte, andwhether the second peak corresponds to the first analyte or the secondanalyte, based on either an area or a height of the first peak and thesecond peak, and the first predetermined calibrant ratio. A firstanalyte time interval is assigned based on one of the first retentiontime and second retention time that corresponds to the first analyte. Asecond analyte time interval is assigned based on one of the firstretention time and second retention time that corresponds to the secondanalyte. A second diluted standard solution is formed by mixing thestandard material and the diluent. The second diluted standard solutionincludes the first analyte having a third diluted calibrantconcentration, and the second analyte having a fourth diluted calibrantconcentration. The second diluted standard solution is injected into thechromatographic separator. Next, the second diluted standard solutionseparated in the chromatographic separator. A third peak and a fourthpeak are measured with the detector. The third peak and the fourth peakhave a third retention time and a fourth retention time, respectively.Automatically identifying that the third peak corresponds to the firstanalyte where the third retention time falls within the first analytetime interval or the second analyte where the third retention time fallswithin the second analyte time interval. Automatically identifying thatthe fourth peak corresponds to the first analyte where the fourthretention time falls within the first analyte time interval or thesecond analyte where the fourth retention time falls within the secondanalyte time interval. Automatically calculating a first calibrationequation for the first analyte based on an area or a height of two ofthe automatically identified peaks that correspond to the first analyte,the two peaks selected from the group consisting of the first peak, thesecond peak, the third peak, and the fourth peak; the first and thirddiluted calibrant concentrations. Automatically calculating a secondcalibration equation for the second analyte based on an area or a heightof two of the automatically identified peaks that correspond to thesecond analyte, the two peaks selected from the group consisting of thefirst peak, the second peak, the third peak, and the fourth peak; andthe second and fourth diluted calibrant concentrations.

In a second method embodiment of calibrating a chromatography system, itincludes at least two automatic identifications step based on a firstpredetermined calibrant ratio. This second method is well suited forcalibrating analyte ions that are strongly dissociated.

The second method includes forming a first diluted standard solution bymixing a standard material and a diluent. The first diluted standardsolution includes a first analyte having a first diluted calibrantconcentration, and a second analyte having a second diluted calibrantconcentration, in which the first diluted standard solution isconfigured to have a first predetermined calibrant ratio. The firstdiluted standard solution is injected into a chromatographic separator.The first diluted standard solution is separated in the chromatographicseparator. A first peak and a second peak are measured with a detector.The first peak and second peak have a first retention time and a secondretention time, respectively. Automatically identifying whether thefirst peak corresponds to the first analyte or the second analyte, andwhether the second peak corresponds to the first analyte or the secondanalyte, based on either an area or a height of the first peak and thesecond peak, and the first predetermined calibrant ratio. A seconddiluted standard solution is formed by mixing the standard material andthe diluent. The second diluted standard solution includes the firstanalyte having a third diluted calibrant concentration, and the secondanalyte having a fourth diluted calibrant concentration. The seconddiluted standard solution is injected into the chromatographicseparator. Next, the second diluted standard solution separated in thechromatographic separator. A third peak and a fourth peak are measuredwith the detector. The third peak and the fourth peak have a thirdretention time and a fourth retention time, respectively. Automaticallyidentifying whether the third peak corresponds to the first analyte orthe second analyte, and whether the fourth peak corresponds to the firstanalyte or the second analyte, based on either an area or a height ofthe third peak and the fourth peak, and the first predeterminedcalibrant ratio. Automatically calculating a first calibration equationfor the first analyte based on an area or a height of two of theautomatically identified peaks that correspond to the first analyte, thetwo peaks selected from the group consisting of the first peak, thesecond peak, the third peak, and the fourth peak; the first and thirddiluted calibrant concentrations. Automatically calculating a secondcalibration equation for the second analyte based on an area or a heightof two of the automatically identified peaks that correspond to thesecond analyte, the two peaks selected from the group consisting of thefirst peak, the second peak, the third peak, and the fourth peak; andthe second and fourth diluted calibrant concentrations.

In regards to the first and second method, the first calibrationequation can include a first calibration slope and a first y-interceptand the second calibration equation can include a second calibrationslope and second y-intercept.

In regards to the first and second method, the first and secondcalibration equations can be in a polynomial form. The calculations forthe polynomial equations can require a third calibration point. Thismethod includes forming a third diluted standard solution by mixing astandard material and a diluent. The third diluted standard solutionincludes the first analyte having a fifth diluted calibrantconcentration, and the second analyte having a sixth diluted calibrantconcentration. The third diluted standard solution is injected into thechromatographic separator. The third diluted standard solution isseparated in the chromatographic separator. A fifth peak and a sixthpeak are measured with the detector. The fifth peak and the sixth peakhave a fifth retention time and a sixth retention time, respectively.Automatically identifying that the fifth peak corresponds to the firstanalyte where the fifth retention time falls within the first analytetime interval or the second analyte where the fifth retention time fallswithin the second analyte time interval. Automatically identifying thatthe sixth peak corresponds to the first analyte where the sixthretention time falls within the first analyte time interval or thesecond analyte where the sixth retention time falls within the secondanalyte time interval. Automatically calculating the first calibrationequation for the first analyte based on an area or a height of three ofthe automatically identified peaks that correspond to the first analyte,the three peaks selected from the group consisting of the first peak,the second peak, the third peak, the fourth peak, the fifth peak, andthe sixth peak, and the first, third, and fifth diluted calibrantconcentrations. Automatically calculating a second calibration equationfor the second analyte based on an area or a height of three of theautomatically identified peaks that correspond to the second analyte,the three peaks selected from the group consisting of the first peak,the second peak, the third peak, the fourth peak, the fifth peak, andthe sixth peak, and the second, fourth, and sixth diluted calibrantconcentrations. The first calibration equation includes a firstpolynomial equation, and the second calibration equation includes asecond polynomial equation. More particularly, the first polynomialequation can be a first quadratic equation and the second polynomialequation can be a second quadratic equation.

In regards to any of the above methods, the first and second dilutedsolutions can be prepared using one or more pumps where the standardmaterial includes a standard solution. The forming of the first dilutedstandard solution includes pumping a first aliquot of the standardsolution and a second aliquot of the diluent into the junction. Thestandard solution includes the first analyte having a first calibrantconcentration, and the second analyte having a second calibrantconcentration. The first aliquot and the second aliquot can be mixed toform the first diluted standard solution. The forming of the seconddiluted standard solution includes pumping a third aliquot of thestandard solution and a fourth aliquot of the diluent into the junction.The third aliquot and fourth aliquot can be mixed to form the seconddiluted standard solution. In an embodiment, the pumps can be syringepumps.

In regards to any of the above methods, a user can input a concentrationrange of expected analyte concentrations that includes a lower analyteconcentration and an upper analyte concentration for a particularanalyte. In turn, this method can perform a seamless calibration processthat accounts for a user inputted analyte concentration range to ensurethat the diluted calibrant concentrations span a range similar to therange of expected analyte concentrations. A first concentration rangefor the first analyte can be received before forming the first dilutedstandard solution and the second diluted standard solution. The firstconcentration range can include a lower first analyte concentration andan upper first analyte concentration. Automatically calculating thefirst diluted calibrant concentration based on the upper first analyteconcentration. Automatically setting a first flow rate or a first pumpduration time for the first aliquot and a second flow rate or a secondpump duration time for the second aliquot to form the automaticallycalculated first diluted calibrant concentration. Automaticallycalculating the third diluted calibrant concentration based on the lowerfirst analyte concentration. Automatically setting a third flow rate ora third pump duration time for the third aliquot and a fourth flow rateor a fourth pump duration time for the fourth aliquot to form theautomatically calculated third diluted calibrant concentration.

A sample containing a first analyte can be analyzed after thechromatography system has been calibrated with any of the above methods.A first sample analysis method includes injecting a first sample intothe chromatographic separator where the first sample includes at least afirst analyte. The first sample is separated in the chromatographicseparator. A seventh peak is measured with the detector that has aseventh retention time. Automatically identifying that the seventh peakcorresponds to the first analyte where the seventh retention time fallswithin the first analyte time interval. Automatically calculating afirst analyte concentration of the first sample based on an area or aheight of the seventh peak and the first calibration equation.

In regards to the first sample analysis methods, a feedback controlmethod can be implemented when the automatically calculated analyteconcentration is too low to be effectively analyzed with presentcalibration parameter. As a result, a recalibration process can beautomatically triggered when such a low analyte concentration ismeasured. This method includes calculating a seventh diluted calibrantconcentration for the first analyte is that is less than theautomatically calculated first analyte concentration when theautomatically calculated first analyte concentration is less than alower first analyte concentration of a first concentration range. Afourth diluted standard solution is formed by mixing the standardsolution and the diluent. The fourth diluted standard solution includesa first analyte having the seventh diluted calibrant concentration. Thefourth diluted standard solution is injected into the chromatographicseparator. The fourth diluted standard solution is separated in thechromatographic separator. An eighth peak is measured with the detectorthat has an eighth retention time. Automatically identifying that theeighth peak corresponds to the first analyte where the eighth retentiontime falls within the first analyte time interval. Automaticallycalculating an adjusted first calibration equation for the first analytebased on an area or a height of the eighth peak and at least one peak ofthe first, second, third, and fourth peaks that corresponds to the firstanalyte, and the seventh diluted calibrant concentration and at leastone of the first, second, third, and fourth diluted calibrantconcentrations that corresponds to the first analyte.

In regards to the first sample analysis method and the above feedbackcontrol method, another feedback control method can be implemented whenthe automatically calculated analyte concentration is too high to beeffectively analyzed with present calibration parameter. As a result, arecalibration process can be automatically triggered when such a highanalyte concentration is measured. This method includes calculating aneighth diluted calibrant concentration for the first analyte is that isgreater than the automatically calculated first analyte concentrationwhen the automatically calculated first analyte concentration is greaterthan an upper first analyte concentration of a first concentrationrange. A fifth diluted standard solution is formed by mixing thestandard solution and the diluent. The fifth diluted standard solutionincludes a first analyte having the eighth diluted calibrantconcentration. The fifth diluted standard solution is injected into thechromatographic separator. The fifth diluted standard solution isseparated in the chromatographic separator. A ninth peak is measuredwith the detector that has a ninth retention time. Automaticallyidentifying that the ninth peak corresponds to the first analyte wherethe ninth retention time falls within the first analyte time interval.Automatically calculating an adjusted first calibration equation for thefirst analyte based on an area or a height of the ninth peak and atleast one peak of the first, second, third, and fourth peaks thatcorresponds to the first analyte, and the eighth diluted calibrantconcentration and at least one of the first, second, third and fourthdiluted calibrant concentrations that corresponds to the first analyte.

A sample containing a first analyte and a second analyte can be analyzedafter the chromatography system has been calibrated with any of theabove methods. A second sample analysis method includes injecting afirst sample into the chromatographic separator. The first sampleincludes a first analyte and a second analyte. The first sample isseparated in the chromatographic separator. An eleventh peak and a tenthpeak is measured with the detector. The eleventh peak has an eleventhretention time and the tenth peak has a tenth retention time.Automatically identifying that the eleventh peak corresponds to thefirst analyte where the eleventh retention time falls within the firstanalyte time interval or the eleventh peak corresponds to the secondanalyte where the eleventh retention time falls within the secondanalyte time interval. Automatically identifying that the tenth peakcorresponds to the first analyte where the tenth retention time fallswithin the first analyte time interval or the tenth peak corresponds tothe second analyte where the tenth retention time falls within thesecond analyte time interval. Automatically calculating a first analyteconcentration of the first sample based on an area or a height of one ofthe automatically identified peaks selected from the group consisting ofthe eleventh peak and the tenth peak that corresponds to the firstanalyte and the first calibration equation. Automatically calculating asecond analyte concentration of the first sample based on an area or aheight of one of the automatically identified peaks selected from thegroup consisting of the eleventh peak and the tenth peak thatcorresponds to the second analyte and the second calibration equation.

In regards to any of the above methods, the first analyte time intervalhas a first upper limit and a first lower limit. The first upper limitbeing one of the first retention time and the second retention timecorresponding to the first analyte plus a first predetermined proportionmultiplied by one of the first retention time and the second retentiontime corresponding to the first analyte. The first lower limit being oneof the third retention time and the fourth retention time correspondingto the first analyte minus a first predetermined proportion multipliedby one of the third retention time and the fourth retention timecorresponding to the first analyte. The first predetermined proportionmay range from about 0.05 to about 0.2.

In regards to any of the above methods, the second analyte time intervalhas a second upper limit and a second lower limit. The second upperlimit being one of the first retention time and the second retentiontime corresponding to the second analyte plus a first predeterminedproportion multiplied by one of the first retention time and the secondretention time corresponding to the second analyte. The first lowerlimit being one of the third retention time and the fourth retentiontime corresponding to the second analyte minus a first predeterminedproportion multiplied by one of the third retention time and the fourthretention time corresponding to the second analyte. The secondpredetermined proportion may range from about 0.05 to about 0.2.

In regards to any of the above methods, the junction may include aT-junction.

In regards to any of the above methods, the mixing may occurs in amixing chamber downstream of the T-junction.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and constitutepart of this specification, illustrate presently preferred embodimentsof the invention, and, together with the general description given aboveand the detailed description given below, serve to explain features ofthe invention (wherein like numerals represent like elements).

FIG. 1 illustrates an embodiment of a chromatography system suitable foruse with the chromatographic methods described herein.

FIG. 2 illustrates a simplified schematic of an automated standardsdilution system in a load position that is in accordance with thechromatography system of FIG. 1.

FIG. 3 illustrates a simplified schematic of the automated standardsdilution system in an inject position that is in accordance with thechromatography system of FIG. 1.

FIG. 4 is a flow chart illustrating an automatic method of calibrating achromatography system with diluted standard solutions.

FIG. 5 is a flow chart illustrating an automatic method of automaticallyidentifying and quantifying the analyte in a chromatography system thathas been calibrated.

FIG. 6 shows a series of simulated chromatograms for a first dilutedstandard solution (A), a second diluted standard solution (B), and asample containing a first analyte (C).

DETAILED DESCRIPTION OF EMBODIMENTS

The following detailed description should be read with reference to thedrawings, in which like elements in different drawings are identicallynumbered. The drawings, which are not necessarily to scale, depictselected embodiments and are not intended to limit the scope of theinvention. The detailed description illustrates by way of example, notby way of limitation, the principles of the invention. This descriptionwill clearly enable one skilled in the art to make and use theinvention, and describes several embodiments, adaptations, variations,alternatives and uses of the invention, including what is presentlybelieved to be the best mode of carrying out the invention. As usedherein, the terms “about” or “approximately” for any numerical values orranges indicate a suitable dimensional tolerance that allows the part orcollection of components to function for its intended purpose asdescribed herein.

In a method embodiment of calibration, a standard solution can be run ina chromatographic instrument that produces a predetermined ratio of atleast two peaks at a predetermined (concentration) level. Thechromatogram from this injection is used to identify the peaks. Theretention times required for future identification of the peaks at thatlocation with the appropriate retention time window can be assigned.Next, a series of diluted standards solution can be prepared and run ina chromatographic instrument where the retention time window is used todetect and identify the peaks in an automated fashion. The dilutedstandard solution is identified based on retention times and not thepredetermined ratio. Under certain conditions where one analyte is afully dissociated acid and anther analyte is a weakly dissociatedanalyte, a diluted standard solution may not have a constantpredetermined ratio of two peaks compared to the standard solution(undiluted). In the above method, the calibration process accounts fordifficulties calibrating and analyzing samples including weaklydissociated species and strongly dissociated species. It should be notedthat conductivity detectors can have a linear response for stronglydissociated species and a curved response (e.g., quadratic) for weaklydissociated species.

The following describes a general chromatography system suitable for usewith the methods of calibration and sample analysis described herein.FIG. 1 illustrates an embodiment of an ion chromatography system 500that includes an autosampler 522, a pump 502, an electrolytic eluentgenerating device 503, a degas assembly 510, an injection valve 512, achromatographic separation device 514, an optional suppressor 515, adetector 516, automated standards dilution system 30, a microprocessor518, and a reader 524 such as an RFID reader. A recycle line 520 may beused to transfer the liquid from an output of detector 516 to aregenerant portion of suppressor 515, and an inlet of degas assembly510. Reader 524 may include a bar code reader or a contact mechanism forreading data using wires.

Pump 502 can be configured to pump a liquid from a liquid source and befluidically connected to electrolytic eluent generating device 503.Electrolytic eluent generating device 503 is configured to generate aneluent such as for example KOH or methanesulfonic acid. Detailsregarding electrolytic eluent generating devices can be found in U.S.Pat. Nos. 6,225,129 and 6,682,701, which are hereby incorporated byreference herein. In an embodiment, a residual gas may be carbondioxide, hydrogen, and oxygen. The gas can be swept out of degasassembly 510 using a recycled liquid via a recycle line 520 that isdownstream of detector 516. Injection valve 512 can be used to inject analiquot of a liquid sample into an eluent stream. Chromatographicseparation device 514 can be used to separate various matrix componentspresent in the liquid sample from the analytes of interest. An output ofchromatographic separation device 514 can be fluidically connected tosuppressor 515, and then to detector 516 to measure the presence of theseparated chemical constituents of the liquid sample.

Suppressor 515 is a device used in ion chromatography to remove theeluent and sample counterions and replace them with regenerant ions. Asa result, the eluent is converted a weakly dissociated form prior toentering the detector. The suppressor allows analyte ions to be detectedwith a conductivity detector with a low background. Furthermore, theanalytes can be converted to the more conductive acid or base form,which enhances the signal, particularly for fully dissociated species.Detail regarding suppressors can be found in U.S. Pat. Nos. 4,999,098;6,328,885; and 8,415,168 which are hereby fully incorporated byreference herein.

Detector 516 may be in the form of ultraviolet-visible spectrometer, afluorescence spectrometer, an electrochemical detector, a conductometricdetector, a charge detector, or a combination thereof. Details regardingthe charge detector that is based on a charged barrier and twoelectrodes can be found in US Pre-Grant Publication No. 20090218238,which is hereby fully incorporated by reference herein. For thesituation where recycle line 520 is not needed, detector 516 may also bein the form of a mass spectrometer or a charged aerosol detector. Thecharged aerosol detector nebulizes the effluent flow and creates chargedparticles that can be measured as a current proportional to the analyteconcentration. Details regarding the charged aerosol detector can befound in U.S. Pat. Nos. 6,544,484; and 6,568,245, which are hereby fullyincorporated by reference herein.

An electronic circuit may include microprocessor 518 and a memoryportion. Microprocessor 518 can be used to control the operation ofchromatography system 500. Microprocessor 518 may either be integratedinto chromatography system 500 or be part of a personal computer thatcommunicates with chromatography system 500. Microprocessor 518 may beconfigured to communicate with and control one or more components ofchromatography system such as autosampler 522, reader 524, pump 502,electrolytic eluent generating device 503, injection valve 512, anddetector 516. Note that chromatography system 500 is a particularmachine used to analyze standard solutions and sample solutions toidentify chemical constituents and the associated concentration values.

An automated standards dilution system can be used to create a varietyof dilutions of a standard solution. Standard solutions may have one ormore analytes where each analyte is at a predetermined concentration. Asillustrated in FIGS. 1 to 3, automated standards dilution system 30includes a concentrate pump 10, a diluent pump 12, a junction 16, and anoptional mixer 20. The output of concentrate pump 10 and diluent pump 12can be inputted into junction 16 via tubings 14 and 18, respectively, tocombine the two liquids, as illustrated in FIGS. 2 and 3. Junction 16may be in the form of a “tee” junction (i.e. T-junction). Whereadditional mixing is needed in addition to that provided by junction 16,an optional mixer 20 may be used before injecting into injection valve512. After the combination of liquids from concentrate pump 10 anddiluent pump 12, the mixed liquid can flow to injection valve 512. Asillustrated in FIGS. 2 and 3, injection valve 512 is in the form of asix-port injection valve. The mixed liquid containing the standardsolution is loaded onto a sample loop 32 with the excess standardsolution going to a waste reservoir 28 in the load position asillustrated in FIG. 2. The sample loop can be injected towardschromatographic separation when valve 512 is changed to the injectposition.

In an embodiment, concentrate pump 10 can pump a standard solution thathas a relatively high concentration of analytes. An example of aconcentrated standard is the combined five anion standard containing 20mg/L fluoride, 30 mg/L chloride, 100 mg/L nitrate, 150 mg/L phosphate,and 150 mg/L sulfate (commercially available from Thermo ScientificDionex part number 057590, Sunnyvale, Calif., USA). Diluent pump 12 canpump a diluent such as, for example, deionized water. For example aconcentrated standard mixture of five anions can be pumped in at a flowrate of 1 μL/min. The diluent pump can be operated at a flow rate of 99μL/min to produce a net flow of a diluted standard solution at 100μL/min. The concentration of the diluted standard solution in this casewould be a 100 fold dilution.

An optional mixer column 20 can be used to mix the standards well beforeinjecting into a loop 32 that is installed in a six port injection valve24. The injection valve has an inlet that is fluidically connected topump 502, and with one or more of the following devices, which are degasassembly 510, electrolytic eluent generating device 503, and autosampler522. The injection valve has an outlet that is fluidically connectedwith chromatographic separation device 514, detector 516, and optionalsuppressor 515.

The flow rate of concentrate pump 10 and diluent pump 12 can be variedso that different dilutions of the standard solution can be generated.In the load position of injection valve 512, loop 32 is loaded with thediluted standard solution as illustrated in FIG. 2. In the injectposition shown in FIG. 2, the contents of loop 32 are injected into anion chromatograph for analysis. The pumps 10 and 12 can be syringe pumpsthat can be driven using a single drive system. The pumps 10 and 12 canalso be in the form of a simple proportioning valve setup that allowsproportioning of the concentrate and diluent at different ratios.Automated standards dilution system 30 can be controlled by a softwareinterface using microprocessor 518.

An automated calibration routine is described that combines the steps ofa) calibrant generation via automated dilution, and b) identificationbased on preset response ratios. This automated calibration routine isconfigured to require a reduced amount user intervention or no userintervention. After calibrating a system, a method of using achromatography system may be used to a) identify peaks of interest basedon retention time b) provide a measured analyte response based on aconcentration curve. In order to get an accurate quantitation, one ormore levels of the standard solutions are chromatographically analyzedduring calibration. Standard solutions may be prepared in an offlinefashion, diluted, and injected into the system for the purpose ofcalibration. Any errors in dilution can impact the calibration curve andhence cause the quantitation to be less accurate.

A method of using an analytical system typically includes a calibrationstep and a sample analysis step. The following will describe a method ofcalibrating a chromatography system in more detail. FIG. 4 is a flowchart illustrating an automatic method 400 of calibrating achromatography system with a standard solution. Method 400 may includeforming a first diluted standard solution (step 402), injecting thefirst diluted standard solution (step 404), separating the first dilutedstandard solution (step 406), measuring first and second peaks of thestandard solution (step 408), automatically identifying whether thefirst peak corresponds to the first analyte or second analyte (step410), automatically identifying whether the second peak corresponds tothe first analyte or second analyte (step 412), assigning a firstanalyte time interval (step 414), assigning a second analyte timeinterval (step 416), forming a second diluted standard solution (step418), injecting the second diluted standard solution (step 420),separating the second diluted standard solution (step 422), measuringthird and fourth peaks of the standard solution (step 424),automatically identifying whether the third peak corresponds to thefirst analyte or second analyte (step 426), automatically identifyingwhether the fourth peak corresponds to the first analyte or secondanalyte (step 428), automatically calculating a first calibrationequation for the first analyte (step 430), and automatically calculatinga second calibration equation for the second analyte (step 432).

In step 402, a first diluted standard solution is formed by mixing astandard material and a diluent. The first diluted standard solution mayinclude a first analyte having a first diluted calibrant concentrationand a second analyte having a second diluted calibrant concentration.The first diluted standard solution is configured to have a firstpredetermined calibrant ratio.

A standard material may be in the form of a concentrated liquid solutionor a solid compound. In an embodiment, the standard material can be astandard solution and contains a plurality of analytes. For example, thestandard solution may be a five anion standard containing fluoride,chloride, nitrate, phosphate, and sulfate. The solid compound can be anaggregate of analyte salts. In an embodiment, a manual process can beimplemented to prepare a combination of solid analytes for the standardmaterial. The diluent may be a liquid solution such as deionized water.The standards can be prepared on a weight by weight or volume by volumebasis.

The first predetermined calibrant ratio can be a numerical valueassigned to the first diluted standard solution. The first predeterminedcalibrant ratio can be based on a ratio of a peak area corresponding toa first analyte and a peak area corresponding to the second analyte.Alternatively, a peak height can be used instead of peak area where thefirst predetermined calibrant ratio is based on a ratio of a peak heightcorresponding to a first analyte and a peak height corresponding to thesecond analyte.

Prior to the calibration process 400, the standard material can becharacterized using chromatography to establish a predeterminedcalibrant ratio. For example, a standard solution can include fluorideand chloride where the respective concentrations are adjusted so thatthe measured peak areas on a conductivity detector have a peak arearatio of 2:1, which corresponds to the first predetermined calibrantratio. In this example, the fluoride has an approximately two-foldhigher concentration than chloride. Under certain circumstances, themeasured peak areas are not always proportional based on concentrationvalues because the sensitivity of fluoride and chloride may be differenton a conductivity detector. If this were case, the concentrations offluoride and chloride would be adjusted until the desired peak arearatio was measured. It should be noted that method 400 is not limited toone predetermined calibrant ratio and that multiple predeterminedcalibrant ratios can be used for standard solutions containing multipleanalytes. For simplicity, method 400 will be described with two analytesand one predetermined calibrant ratio. It should be noted that method400 is not limited to only two analytes and that one having ordinaryskill in the art would be able to apply method 400 to more than twoanalytes.

The following will describe an automated method of preparing a dilutedstandard solution where the standard material is a standard solution.The forming of the first diluted standard solution includes pumping afirst aliquot of the standard solution into a junction and pumping asecond aliquot of a diluent into the junction. The first aliquot and thesecond aliquot can be mixed in the junction to form the first dilutedstandard solution. The junction may be in the form of a tee junctionwhere the first aliquot and second aliquot are inputted into tworespective inlets of the tee junction and outputted as a mixture. Amixer may be used downstream of the tee junction for certaincircumstances where more mixing may be needed. In an embodiment, twosyringe pumps may be used to pump the first and second aliquots into thejunction. The pumping of the two aliquots may occur simultaneously ormay occur separately in time. The aliquots can be provided by a flowingstream into the junction or a volume of liquid. Where the two pumpsoperate simultaneously, the flow rates of the two pumps can be set sothat the appropriate magnitudes of the two aliquots are generated toprovide the diluted standard solution. Where the two pumps do notoperate simultaneously, the flow rates of the two pumps can be set bythe outputted volume so that the appropriate magnitudes of the twoaliquots are generated to provide the diluted standard solution.

Once the first diluted standard solution is prepared, it is injectedinto a chromatographic separator in step 404. In an embodiment, thechromatographic separator may be an ion exchange column and the analytesmay be anions. The first diluted standard solution is separated in thechromatographic separator in step 406. A first peak and a second peakcan be measured with a detector in step 408. It should be noted that theseparation of the sample is a physical transformation of the sample thatseparates the analytes into discrete portions that elute off of thechromatography column.

FIG. 6 illustrates an exemplary chromatogram A of the first dilutedstandard solution showing a detection of a first peak P1 and a secondpeak P2 that have a first retention time t1 and second retention timet2, respectively. Note that the peaks P1 and P2 are depicted as linesfor simplicity and that the peaks can be of other shapes typicallyobserved in chromatograms such as Gaussian.

In step 410, the method 400 includes automatically identifying whetherthe first peak corresponds to the first analyte or the second analyte.Similarly, step 412 automatically identifies whether the second peakcorresponds to the first analyte or the second analyte. This automaticidentification process is based on either an area or a height of thefirst peak and the second peak, and the first predetermined calibrantratio. The first predetermined calibrant ratio [2^(nd) Analyte/1^(st)Analyte] can be multiplied by the first peak area to see if the productvalue corresponds to the second peak area, and if so, the first peakwill correspond to the first analyte and the second peak to the secondanalyte. Similarly, the ratio value can be multiplied by the second peakarea to see if the product value corresponds to the first peak area, andif so, the second peak will correspond to the first analyte and thefirst peak to the second analyte. It should be noted that as describedherein that the analyte identity is calculated without using theretention times.

For example, the first peak area can be 0.25, the second peak area canbe 0.5, and the first predetermined calibrant ratio can be 2:1(acetate:chloride). In an embodiment, the first predetermined calibrantratio×the first peak area equals the second peak area indicating thatthe first peak is chloride and the second peak is acetate (e.g.,2/1×0.25=0.5). In addition, the first predetermined calibrant ratio×thesecond peak area does not equal the first peak area indicating that thefirst peak is not acetate and the second peak is not chloride (e.g.,2/1×0.5≠0:25). In accordance with the exemplary chromatogram A of FIG.6, the first peak P1 would be the first analyte chloride and the secondpeak P2 would be the second analyte acetate. Step 410 may includeevaluate multiple permutations of pairs of peak areas usingpredetermined calibrant ratios to identify analytes. The automaticidentification process of steps 410 and 412 is similar to filed U.S.patent application Ser. No. 13/834,883 [Attorney Docket No.16633US1/NAT], filed on Mar. 15, 2013, which is hereby fullyincorporated by reference herein.

In step 414, a first analyte time interval can be assigned based on oneof the first retention time and second retention time that correspondsto the first analyte. Similarly, in step 416, a second analyte timeinterval can be assigned based on one of the first retention time andsecond retention time that corresponds to the second analyte. In anembodiment, a user can assign a first analyte time interval to theappropriate retention time corresponding to the first analyte. Asoftware interface can be used so that a user can input a first analytetime interval that includes a first upper limit and a first lower limitwhere the retention time corresponding to the first analyte is inbetween the first upper limit and the first lower limit. A user may wantto manually set the first analyte time interval to exclude co-elutingpeaks. In another embodiment, the first upper limit can be the retentiontime corresponding to the first analyte plus a first predeterminedproportion multiplied by this retention time and the first lower limitbeing the retention time corresponding to the first analyte minus thefirst predetermined proportion multiplied by this retention time. Thesecond upper limit can be the retention time corresponding to the secondanalyte plus a second predetermined proportion multiplied by thisretention time and the second lower limit being the retention timecorresponding to the second analyte time minus the second predeterminedproportion multiplied by this retention time. The first predeterminedproportion and second predetermined proportion may each range from about0.05 to about 0.2.

As an example, the retention time corresponding to the first analyte canbe one minute and the first predetermined proportion can be 0.1. Thefirst lower limit would be 0.9 minutes (1−(0.1×1)) and the first upperlimit would be 1.1 minutes (1+(0.1×1)).

In another embodiment, the first upper limit and the first lower limitcan be defined by having a fixed window such as 0.1 minutes. In thisexample, the window would be plus or minus 0.05 minutes of the firstretention time.

As an example, where it is assumed that the first peak is the firstanalyte and the second peak is the second analyte, chromatogram A ofFIG. 6 illustrates a first analyte time interval t_(AN1) and a secondanalyte time interval t_(AN2), which are based on the first retentiontime t1 and the second retention time t2, respectively. Moreparticularly, the first analyte time interval t_(AN1) can have a firstupper limit t_(UL1) and a first lower limit t_(LL1), as illustrated inFIG. 6. The first upper limit t_(UL1) can be the first retention time t1plus a first predetermined proportion of the first retention time t1 andthe first lower limit t_(LL1) can be the first retention time t1 minusthe first predetermined proportion of the first retention time t1.Similar to the first analyte time interval t_(AN1), the second analytetime interval t_(AN2) can have a second upper limit t_(UL2) and a secondlower limit t_(LL2), as illustrated in FIG. 6. The second upper limitt_(UL2) can be the second retention time t2 plus a second predeterminedproportion of the second retention time t2 and the second lower limitt_(LL2) can be the second retention time t2 minus the secondpredetermined proportion of the second retention time t2.

In step 418, a second diluted standard solution is formed by mixing thestandard material and the diluent. The second diluted standard solutionmay include a first analyte having a third diluted calibrantconcentration and a second analyte having a fourth diluted calibrantconcentration. The first diluted standard solution is configured to havea first predetermined calibrant ratio. In an embodiment, the firstdiluted calibrant concentration is higher than the third dilutedcalibrant concentration for the first analyte, and the second dilutedcalibrant concentration is higher than the fourth diluted calibrantconcentration for the second analyte.

Once the second diluted standard solution is prepared, it is injectedinto a chromatographic separator in step 420. The second dilutedstandard solution is separated in the chromatographic separator in step422. A third peak and a fourth peak can be measured with a detector instep 424. The first peak and the second peak can have a third retentiontime and a fourth retention time, respectively.

As an example, where it is assumed that the first peak is the firstanalyte and the second peak is the second analyte, chromatogram B ofFIG. 6 illustrates a chromatographic separation of the second dilutedstandard solution that results in a third peak P3 and a fourth peak P4that have a third retention time t3 and a fourth analyte retention timet4, respectively.

Step 426 automatically identifies that the third peak corresponds to thefirst analyte where the third retention time falls within the firstanalyte time interval or the second analyte where the third retentiontime falls within the second analyte time interval. Similarly, step 428automatically identifies that the fourth peak corresponds to the firstanalyte where the fourth retention time falls within the first analytetime interval or the second analyte where the fourth retention timefalls within the second analyte time interval. It should be noted thatidentification steps 410 and 412 can be based on the first predeterminedcalibrant ratio whereas identification steps 426 and 428 can be based onfirst and second analyte time intervals. Applicant unexpectedlydiscovered that the combination of using a predetermined calibrant ratio(in steps 410 and 412) and then time intervals (in steps 426 and 428)provided an advantage when analyzing samples that contain at least oneanalyte that is weakly dissociated such as, for example, acetate. Weaklydissociated ions exist mainly in a non-ionized form. Under certaincircumstances, a conductivity detector provides a non-linear response toanalytes that are weakly dissociated. As a result, the predeterminedcalibrant ratio may not be constant for both the first and seconddiluted calibrant solutions. In contrast, samples that contain onlyfully or strongly dissociated analytes such as, for example, chloridehave a constant predetermined calibrant ratio for both the first andsecond diluted calibrant solution. Strongly dissociated ions existmainly in an ionized form. When all of the analytes are fullydissociated, steps 426 and 428 can be similar to steps 410 and 412 wherethe identification is also based on the predetermined calibrant ratio.

As an example, where it is assumed that the first peak is the firstanalyte and the second peak is the second analyte, chromatogram B ofFIG. 6 illustrates that the third retention time t3 falls within thefirst analyte time interval t_(AN1) and that the fourth retention timet4 falls within the second analyte time interval t_(AN2). Note that aretention time falls within an analyte time interval where the retentiontime is greater than t_(LL) and less than the t_(UL).

Step 430 automatically calculates a first calibration equation for thefirst analyte based on an area or a height of two of the automaticallyidentified peaks that correspond to the first analyte, the first dilutedcalibrant concentration, and the third diluted calibrant concentration.The two peaks can be selected from the group consisting of the firstpeak, the second peak, the third peak, and the fourth peak (measured insteps 408 and 424). Similarly, step 432 automatically calculates asecond calibration equation for the second analyte based on an area or aheight of two of the automatically identified peaks that correspond tothe second analyte, the second diluted calibrant concentration, and thefourth diluted calibrant concentration. The two peaks can be selectedfrom the group consisting of the first peak, the second peak, the thirdpeak, and the fourth peak (measured in steps 408 and 424).

In an embodiment, the first and second calibration equation can becalculated using linear regression. For this linear fit, the firstcalibration equation includes a first calibration slope and a firsty-intercept and the second calibration equation includes a secondcalibration slope and second y-intercept.

For the situation where the calibration results in a non-linearresponse, the first and second calibration equation can be a first andsecond polynomial equation. A third calibration point is typicallyneeded for calculating a polynomial equation so method 400 can furtherinclude forming another diluted standard solution by mixing a standardmaterial and a diluent.

In an embodiment, method 400 may include a feature where a user caninput the relevant concentration range of the analyte to be measured.This inputting of the concentration range allows the method toautomatically set the appropriate concentrations for calibrating thesystem. For instance, calibration concentrations are typically slightlyless than the lowest expected analyte concentration and slightly higherthan the highest expected analyte concentration. Before forming thefirst diluted standard solution and the second diluted standard solutionin steps 402 and 418, a user can input a first concentration range intoa user interface of a chromatography software system. The concentrationrange represents the lowest and highest analyte concentrations expectedto be found by the user in a sample containing the analyte. Thisinputted first concentration range is received by the user interfacesoftware. The first concentration range includes a lower first analyteconcentration and an upper first analyte concentration.

The first diluted calibrant concentration can be automaticallycalculated based on the upper first analyte concentration. Next, a) afirst flow rate or a first pump duration time for the first aliquot andb) a second flow rate or a second pump duration time for the secondaliquot can be automatically set to form the automatically calculatedfirst diluted calibrant concentration.

Similarly, the third diluted calibrant concentration can beautomatically calculated the based on the lower first analyteconcentration. Next, c) a third flow rate or a third pump duration timefor a third aliquot and d) a fourth flow rate or a fourth pump durationtime for the fourth aliquot can be automatically set to form theautomatically calculated third diluted calibrant concentration.

Now that automatic calibration processes have been described, thefollowing will describe methods for analyzing a sample. In anembodiment, a method 450 includes a step 452 of injecting a first sampleinto the chromatographic separator, as shown in FIG. 5. The first sampleincludes at least a first analyte. The first sample is separated in thechromatographic separator as shown in step 454. A sample analyte peak ismeasured with the detector as shown in step 456. The sample analyte peakmay also be referred to as a seventh peak that has a seventh retentiontime. Step 458 automatically identifies that the seventh peakcorresponds to the first analyte where the seventh retention time fallswithin the first analyte time interval. Step 460 automaticallycalculates a first analyte concentration of the first sample based on anarea or a height of the seventh peak and the first calibration equation.

As an example, chromatogram C of FIG. 6 illustrates a chromatogram of afirst sample that results in a seventh peak, having a seventh retentiontime t7. The seventh retention time t7 falls within the first analytetime interval t_(AN1) indicating that the sample includes the firstanalyte. The height or area of the seventh peak can be used to calculatethe concentration of the first analyte using the first calibrationequation.

Under certain circumstances, the automatically calculated first analyteconcentration can be less than the lower first analyte concentration ofthe first concentration range. In such a case, a feedback controlmechanism can be implemented where the previously set lower firstanalyte concentration is adjusted to be lower than the automaticallycalculated first analyte concentration. In turn, the calibration isredone with a fourth diluted standard solution. A seventh dilutedcalibrant concentration can be calculated for the first analyte that isless than the automatically calculated first analyte concentration. Thefourth diluted standard solution can be formed by mixing the standardsolution and the diluent. The fourth diluted standard solution includesa first analyte having the seventh diluted calibrant concentration. Thefourth diluted standard solution can be injected into thechromatographic separator. The fourth diluted standard solution can beseparated in the chromatographic separator. An eighth peak can bemeasured, with the detector, that has an eighth retention time.Automatically identifying that the eighth peak corresponds to the firstanalyte where the eighth retention time falls within the first analytetime interval. Automatically calculating an adjusted first calibrationequation for the first analyte based on an area or a height of theeighth peak corresponding to the new lower first analyte concentrationand at least one peak of the first, second, third, and fourth peaks thatcorresponds to the first analyte, and the seventh diluted calibrantconcentration and at least one of the first, second, third, and fourthdiluted calibrant concentrations that corresponds to the first analyte.

Under certain circumstances, the automatically calculated first analyteconcentration can be greater than the upper first analyte concentrationof the first concentration range. In such a case, a feedback controlmechanism can be implemented where the previously set upper firstanalyte concentration is adjusted to be higher than the automaticallycalculated first analyte concentration. In turn, the calibration isredone with a fifth diluted standard solution. An eighth dilutedcalibrant concentration can be calculated for the first analyte that isgreater than the automatically calculated first analyte concentration.The fifth diluted standard solution can be formed by mixing the standardsolution and the diluent. The fifth diluted standard solution includes afirst analyte having an eighth diluted calibrant concentration. Thefifth diluted standard solution can be injected into the chromatographicseparator. The fifth diluted standard solution can be separated in thechromatographic separator. A ninth peak can be measured, with thedetector, that has a ninth retention time. Automatically identifyingthat the ninth peak corresponds to the first analyte where the ninthretention time falls within the first analyte time interval.Automatically calculating an adjusted first calibration equation for thefirst analyte based on an area or a height of the ninth peakcorresponding to the new upper first analyte concentration and at leastone peak of the first, second, third, and fourth peaks that correspondsto the first analyte, and the eight diluted calibrant concentration andat least one of the first, second, third, and fourth diluted calibrantconcentrations that corresponds to the first analyte.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be apparent to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. While the invention hasbeen described in terms of particular variations and illustrativefigures, those of ordinary skill in the art will recognize that theinvention is not limited to the variations or figures described. Inaddition, where methods and steps described above indicate certainevents occurring in certain order, those of ordinary skill in the artwill recognize that the ordering of certain steps may be modified andthat such modifications are in accordance with the variations of theinvention. Additionally, certain of the steps may be performedconcurrently in a parallel process when possible, as well as performedsequentially as described above. Therefore, to the extent there arevariations of the invention, which are within the spirit of thedisclosure or equivalent to the inventions found in the claims, it isthe intent that this patent will cover those variations as well.

What is claimed is:
 1. A method of calibrating a chromatography system,the method comprising: forming a first diluted standard solution bymixing a standard material and a diluent, the first diluted standardsolution comprising: a first analyte having a first diluted calibrantconcentration, and a second analyte having a second diluted calibrantconcentration, in which the first diluted standard solution isconfigured to have a first predetermined calibrant ratio; injecting thefirst diluted standard solution into a chromatographic separator;separating the first diluted standard solution in the chromatographicseparator; measuring, with a detector, a first peak and a second peakthat have a first retention time and a second retention time,respectively; and automatically identifying whether the first peakcorresponds to the first analyte or the second analyte, and whether thesecond peak corresponds to the first analyte or the second analyte,based on either an area or a height of the first peak and the secondpeak, and the first predetermined calibrant ratio; forming a seconddiluted standard solution by mixing the standard material and thediluent, the second diluted standard solution comprising: the firstanalyte having a third diluted calibrant concentration, and the secondanalyte having a fourth diluted calibrant concentration; injecting thesecond diluted standard solution into the chromatographic separator;separating the second diluted standard solution in the chromatographicseparator; measuring, with the detector, a third peak and a fourth peakthat have a third retention time and a fourth retention time,respectively; automatically identifying whether the third peakcorresponds to the first analyte or the second analyte, and whether thefourth peak corresponds to the first analyte or the second analyte,based on either an area or a height of the third peak and the fourthpeak, and the first predetermined calibrant ratio; automaticallycalculating a first calibration equation for the first analyte based onan area or a height of two of the automatically identified peaks thatcorrespond to the first analyte, the two peaks selected from the groupconsisting of the first peak, the second peak, the third peak, and thefourth peak, and the first and third diluted calibrant concentrations;and automatically calculating a second calibration equation for thesecond analyte based on an area or a height of two of the automaticallyidentified peaks that correspond to the second analyte, the two peaksselected from the group consisting of the first peak, the second peak,the third peak, and the fourth peak, and the second and fourth dilutedcalibrant concentrations.
 2. The method of claim 1, in which the firstcalibration equation comprises a first calibration slope and a firsty-intercept and the second calibration equation comprises a secondcalibration slope and a second y-intercept.
 3. The method of claim 1further comprising: assigning a first analyte time interval based on oneof the first retention time and the second retention time thatcorresponds to the first analyte; assigning a second analyte timeinterval based on one of the first retention time and the secondretention time that corresponds to the second analyte; injecting a firstsample into the chromatographic separator, the first sample comprisingat least a first analyte; separating the first sample in thechromatographic separator; measuring, with the detector, a fifth peakthat has a fifth retention time; automatically identifying that thefifth peak corresponds to the first analyte where the fifth retentiontime falls within the first analyte time interval; and automaticallycalculating a first analyte concentration of the first sample based onan area or a height of the fifth peak and the first calibrationequation.
 4. The method of claim 3 further comprising: calculating afifth diluted calibrant concentration for the first analyte that is lessthan the automatically calculated first analyte concentration when theautomatically calculated first analyte concentration is less than alower first analyte concentration of a first concentration range;forming a third diluted standard solution by mixing the standardsolution and the diluent, the third diluted standard solution comprisinga first analyte having the fifth diluted calibrant concentration;injecting the third diluted standard solution into the chromatographicseparator; separating the third diluted standard solution in thechromatographic separator; measuring, with the detector, a sixth peakthat has a sixth retention time; automatically identifying that thesixth peak corresponds to the first analyte where the sixth retentiontime falls within the first analyte time interval; and automaticallycalculating an adjusted first calibration equation for the first analytebased on an area or a height of the sixth peak and at least one peak ofthe first, second, third, and fourth peaks that corresponds to the firstanalyte, and the fifth diluted calibrant concentration and at least oneof the first, second, third, and fourth diluted calibrant concentrationsthat corresponds to the first analyte.
 5. The method of claim 1, inwhich the standard material includes a standard solution, the forming ofthe first diluted standard solution comprises: pumping a first aliquotof the standard solution into a junction, the standard solutioncomprising: the first analyte having the first calibrant concentration,and the second analyte having the second calibrant concentration;pumping a second aliquot of the diluent into the junction; and mixingthe first aliquot and the second aliquot to form the first dilutedstandard solution; and the forming of the second diluted standardsolution comprises: pumping a third aliquot of the standard solutioninto the junction; pumping a fourth aliquot of the diluent into thejunction; and mixing the third aliquot and fourth aliquot to form thesecond diluted standard solution.
 6. The method of claim 5 furthercomprising: before forming the first diluted standard solution and thesecond diluted standard solution, receiving a first concentration rangefor the first analyte, the first concentration range including a lowerfirst analyte concentration and an upper first analyte concentration;automatically calculating the first diluted calibrant concentrationbased on the upper first analyte concentration; automatically setting afirst flow rate or a first pump duration time for the first aliquot anda second flow rate or a second pump duration time for the second aliquotto form the automatically calculated first diluted calibrantconcentration; automatically calculating the third diluted calibrantconcentration based on the lower first analyte concentration; andautomatically setting a third flow rate or a third pump duration timefor the third aliquot and a fourth flow rate or a fourth pump durationtime for the fourth aliquot to form the automatically calculated thirddiluted calibrant concentration.
 7. The method of claim 6, in which thejunction comprises a T-junction.
 8. The method of claim 7, in which themixing occurs in a mixing chamber downstream of the T-junction.